Roles of Glycoslyation in Notch Signaling

描述(申请人提供)：Notch EGF重复序列上的葡聚糖是Notch信号的关键调节器，因此控制许多细胞类型的发育命运。结构性和缺陷的Notch信号都会导致疾病和癌症。因此，确定影响Notch信号的所有因素是至关重要的。Pofut1编码将O-岩藻糖转移到Notch的蛋白质O-岩藻糖基转移酶，它的失活导致Notch信号的严重中断。Pofut1需要底物GDP-岩藻糖，它必须运输到分泌途径中。缺乏已知的GDP-岩藻糖转运体SLc35c1和推定的GDP-岩藻糖转运体SLc35c2的小鼠表现出相对轻微的Notch信号缺陷，揭示了另一个对Notch信号转运体至关重要的新的GDP-岩藻糖转运体的存在。具体目标1将确定哺乳动物Notch信号所需的GDPkey-岩藻糖转运体(S)。我们将使用SLc35c1/slc35c2双基因敲除突变的成纤维细胞来分离哺乳动物最佳Notch信号所需的Gdp-岩藻糖转运蛋白(S)，确定它们在分泌途径中的位置，并建立Gdp-岩藻糖转运蛋白或转运蛋白伴侣的活性。具体目标2将确定Lfng、Mfng和Rfng糖基转移酶在Notch上将N-乙酰氨基葡萄糖(GlcNAc)转移到O-岩藻糖的单独和互补功能，从而调节Notch信号。这三项都是T细胞最佳发育所必需的。表达单个FNG基因、所有三个FNG基因或不表达FNG基因的小鼠将被用来揭示每个FNG在调节Notch1信号和决定T细胞命运方面的结构/功能关系。这将确定必须被O-岩藻糖修饰的Notch1的关键EGF重复序列，以及这些O-岩藻糖在T细胞发育过程中的结构。第三个目标将集中在Notch EGF重复序列-O-GlcNAc的新修饰上。我们最近发现了果蝇Notch信号基因和EGF特异性O-GlcNAc转移酶Eogt之间的遗传相互作用，并表明O-GlcNAc修饰Notch1 EGF重复序列是最佳配体诱导的HeLa细胞Notch1激活所必需的。具体目标3将确定O-GlcNAc对哺乳动物细胞和小鼠中Notch1信号的重要性。失去O-GlcNAc对Notch受体在细胞表面的转运和稳定性的影响，对于Notch配体结合和在共培养分析中对Notch信号的影响，将被定义。我们将在只表达一个Notch1拷贝的Eogt缺失小鼠中研究胚胎发育和T和B细胞发育过程中的Notch信号缺陷。在果蝇和哺乳动物Notch之间保守的四个O-GlcNAc位点将发生突变，以确定O-GlcNAc的特定功能。合并的结果将揭示重要的新见解，Notch信号是如何被多糖调控的。

DESCRIPTION (provided by applicant): Glycans on Notch EGF repeats are critical regulators of Notch signaling and thus control the developmental fate of many cell types. Both constitutive and defective Notch signaling lead to disease and cancer. Therefore, it is critically important to determine all factors that contribute to robust Notch signaling. Inactivation of Pofut1, which encodes the protein O-fucosyltransferase that transfers O-fucose to Notch, causes severe disruption of Notch signaling. Pofut1 requires the substrate GDP-fucose that must be transported into the secretory pathway. Mice lacking the known GDP-fucose transporter Slc35c1, and the putative GDP-fucose transporter Slc35c2, exhibit comparatively mild Notch signaling defects, revealing the existence of an additional, novel GDP-fucose transporter essential for Notch signaling. Specific Aim 1 will identify the GDP key-fucose transporter(s) required for Notch signaling in mammals. We will use Slc35c1/Slc35c2 double knockout mutant fibroblasts to isolate the GDP-fucose transporter(s) required for optimal Notch signaling in mammals, define their location in the secretory pathway and establish GDP-fucose transport or transporter chaperone activity. Specific Aim 2 will identify individual and complementary functions of Lfng, Mfng and Rfng glycosyltransferases that transfer N-acetylglucosamine (GlcNAc) to O-fucose on Notch, and thereby regulate Notch signaling. All three are necessary for optimal T cell development. Mice expressing a single Fng gene, all three Fng genes or no Fng genes will be used to reveal structure/function relationships for each Fng in regulating Notch1 signaling and determining T cell fates. This will identify critical EGF repeats of Notch1 that must be modified by O-fucose glycans, and the structure of those O-fucose glycans during T cell development. The third aim will focus on a new modification of Notch EGF repeats - O-GlcNAc. We have recently identified genetic interactions between Drosophila Notch signaling genes and the EGF- specific O-GlcNAc-transferase Eogt, and shown that modification of Notch1 EGF repeats by O-GlcNAc is required for optimal ligand-induced activation of Notch1 in HeLa cells. Specific Aim 3 will determine how O-GlcNAc is important for Notch1 signaling in mammalian cells and in the mouse. The effects of the loss of O-GlcNAc on Notch receptor trafficking to, and stability at, the cell surface, for Notch ligand binding, and for Notch signalig in co-culture assays, will be defined. We will investigate Notch signaling defects during embryogenesis and T and B cell development in Eogt null mice expressing only one copy of Notch1. Four O-GlcNAc sites that are conserved between Drosophila and mammalian Notch will be mutated to identify specific functions for O-GlcNAc. The combined results will reveal important new insights into how Notch signaling is regulated by glycans.